A Comparison of In-House Real-Time LAMP Assays with a Commercial Assay for the Detection of Pathogenic Bacteria

Molecular detection of bacterial pathogens based on LAMP methods is a faster and simpler approach than conventional culture methods. Although different LAMP-based methods for pathogenic bacterial detection are available, a systematic comparison of these different LAMP assays has not been performed. In this paper, we compared 12 in-house real-time LAMP assays with a commercialized kit (Isothermal Master Mix) for the detection of Listeria monocytogenes, Salmonella spp, Staphylococcus aureus, Escherichia coli O157, E. coli O26, E. coli O45, E. coli O103, E. coli O111, E. coli O121, E. coli O145 and Streptococcus agalactiae. False-positive results were observed in all 12 in-house real-time LAMP assays, while all the negative controls of Isothermal Master Mix remained negative after amplification. The detection limit of Isothermal Master Mix for Listeria monocytogenes, Salmonella spp, Staphylococcus aureus, Escherichia coli O157, E. coli O26, E. coli O45, E. coli O103, E. coli O111, E. coli O121 and Streptococcus agalactiae was 1 pg, whereas the sensitivity of the commercialized kit for E. coli O145 was 100 pg. In conclusion, the 12 in-house real-time LAMP assays were impractical to use, while the commercialized kit Isothermal Master Mix was useful for the detection of most bacterial pathogens.


Introduction
Loop-mediated isothermal amplification (LAMP), developed by Notomi et al. in 2000 [1], can specifically, sensitively and rapidly amplify nucleic acids utilizing a DNA polymerase enzyme with high strand displacement activity and two pairs of primers recognizing six independent sequences of a target gene under isothermal conditions. Moreover, Nagamine et al. in 2002 has advanced the method by incorporating forward loop primers that accelerate the LAMP reaction [2]. Due to the cost effectiveness and sensitivity of LAMP, this method has been widely applied to basic medical research in and environmental testing, as well as point-of-care testing and diagnosis of infectious diseases in clinical settings [3]. In addition, LAMP has also been applied for pathogen detection, and has successfully been used to detect Listeria monocytogenes [4], Salmonella, spp [5], Staphylococcus aureus [6], Escherichia coli O157 [7,8], E. coli O26 [7], E. coli O45 [7], E. coli O103 [7], E. coli O111 [7], E. coli O121 [7], Streptococcus agalactiae [9], Actinobacillus actinomycetemcomitans [10], Mycobacterium tuberculosis [11], and Streptococcus pneumonia [12]. However, a systematic evaluation of these different LAMP assays has not been performed.

Non-Specific Amplification
After the negative controls without genomic DNA were kept at the described temperature for 50 min, false-positive results were observed among all 12 in-house real-time LAMP assays for detection of L. monocytogenes, Salmonella, Staphylococcus aureus, E. coli O157, E. coli O26, E. coli O45, E. coli O103, E. coli O111, E. coli O121 and Streptococcus agalactiae, while all negative controls of the commercial kit Isothermal Master Mix remained negative after amplification at 65 °C for 50 min, as indicated in Table 1. The cross pollution caused by DNA templates as well as amplified products of the LAMP reactions had been excluded, therefore, the non-specific amplification of 12 in-house real-time LAMP assays could be caused by primer dimers, in contrast, the commercial assay can be free of such non-specific amplification.

Sensitivity of the Commercial Assay
Because of the serious non-specific amplification, determining the detection limits of the 12 in-house real-time LAMP assays was of no practical significance, and only the sensitivity of the commercial Isothermal Master Mix kit for pathogenic bacteria detection was determined.
As Table 2 indicates, the detection limit of Isothermal Master Mix for Listeria monocytogenes, Salmonella spp, Staphylococcus aureus, Escherichia coli O157, E. coli O26, E. coli O45, E. coli O103, E. coli O111, E. coli O121 and Streptococcus agalactiae was 1 pg. However, all reactions with serial dilutions of E. coli O145 DNA template ranging from 0.1-100 pg were negative, and then we repeated the experiment, one of four reactions with 100 pg E. coli O145 DNA template was positive (data not shown), therefore, the LAMP assay with the commercial kit Isothermal Master may not suitable for detection of some pathogenic bacteria because of the low sensitivity.

LAMP Primers
The

Determination of Non-Specific Amplification
After the LAMP primers were synthesized by Sangon Biotech Co., Ltd (Shanghai, China), the non-specific amplification of the 12 in-house real-time LAMP assays as well as the commercial Isothermal Master Mix kit were determined via the corresponding negative controls with no genomic DNA. The experiment was performed before DNA extraction, therefore, cross pollution caused by DNA template as well as amplified products of LAMP reactions can be avoided, and the cause of false-positive results can be objectively judged.
The reaction mixtures and reaction conditions of the 12 in-house real-time LAMP assays were as described [4][5][6][7][8][9]. The 25 µL in house reaction system contained 1.  [13]. The reactions of negative controls were held at 65 °C for 50 min with real-time fluorescence monitoring, and each experiment was repeated three times.

Bacteria Strains and DNA Extraction
Tweleve strains used for this study (

Conclusions
Twelve reported in-house LAMP assays for the detection of pathogenic bacteria [4][5][6][7][8][9] have been compared with the commercialized Isothermal Master Mix kit in this study. False-positive results have been observed among all 12 in-house real-time LAMP assays, and it can be concluded from our experiments that the non-specific amplification is caused by primer dimers. It is difficult to avoid primer dimers and non-specific amplification when multiple sets of primers are used in the in-house LAMP assays. This is especially true when the concentrations of primers, Mg 2+ , dNTPs and DNA polymerase in reaction mixtures are as high as those used in real-time PCR [14]. The concentrations of these four factors must be strictly controlled to avoid non-specific amplification in real-time PCR as well as LAMP reactions [15].
False-positive results have not been found in real-time LAMP assays with the commercialized Isothermal Master Mix kit. It is speculated that the Isothermal Master Mix kit may contain some enhancing agents, which can decrease the non-specific amplification. The detection limits for most tested pathogenic bacteria are 1 pg DNA template, and the sensitivity of the commercial assay for E. coli O145 is 100 pg DNA template. In summary, the 12 in-house real-time LAMP assays were impractical for detection of the corresponding pathogenic bacteria, while the commercial Isothermal Master Mix kit was useful for detection of the most pathogenic bacteria.
Project of Xuchang University for Outstanding Young Backbone Teachers, and Training Project for Outstanding Youth Backbone Personnel of Xuchang University.

Author Contributions
Y.W., F.X., W.G. and Y.Z. performed the experiments. D.W. and A.W. wrote the paper. Y.L. designed the experiments.